2.1 Technical Field
The present invention relates to a method of producing osteocyte cell lines in various stages of differentiation. Such cell lines remain stable after more than 20 passages. The osteocyte has a stellate shape with dendritic processes and expresses high level of osteocalcin. More specifically, it provides a method of production for cultured osteocytes of various differentiation stages. Furthermore, it relates to osteocyte cell line, and more specifically cultured osteocyte. The invention also relates to a method for the production of monoclonal antibodies using such cultured osteocytes and further relates to hybridomas and monoclonal antibodies which recognize an osteocyte-specific antigen. Finally the invention relates to a method of screening for modification factors and binding factors for osteocytes.
2.2 Description of Related Art
Bone loss can occur under conditions of disuse or with certain diseases of bone. Examples of bone loss due to disuse include that associated with immobilization and zero gravity. Bone loss can also occur due to estrogen deficiency due to menopause or ovariectomy and also occurs naturally with the aging process.
Osteocytes are the most abundant of the bone cells (approximately 25,000 per mm3 bone or ten times as many osteocytes as osteoblasts) and are found within the mineralized bone matrix (Parfitt, 1977). Because they are buried in the mineralized matrix, they are relatively inaccessible and have been difficult to study in culture in homogeneous populations. It has been suggested that the osteocyte is the most mature or most terminally differentiated form of the osteoblast. However, the properties and functions of osteocytes are poorly understood.
During bone formation, some osteoblasts (osteocyte precursors) are trapped in the forming osteoid tissue while the bone formation front moves on. The trapped or encapsulated cell produces long, slender dendrite-like processes. These processes maintain contact with other osteocytes and with osteoblasts and lining cells on the bone surface (for review, see Aarden et al., 1994). Osteocytes enclosed within osteons appear to be stellate in shape and isolated osteocytes can retain this stellate shape in culture. The formation of cytoplasmic processes by the maturing osteocyte are asynchronous and asymmetrical (Palumbo et al., 1990). The cells produce dendritic processes on the mineralization side before producing processes on the vascular side. Thus the morphology of an osteocyte can range from the stellate or xe2x80x98star-likexe2x80x99 shape to that with extensive cytoplasmic, slender processes longer than the main body of the cell. Osteocytes express a dendritic phenotype both in vivo and in vitro. It has been shown previously that osteocytes express large amounts of osteocalcin.
In addition to their distinctive morphology, osteocytes are now characterized by expression of surface antigens and other markers. Osteocytes strongly express CD44, a transmembrane glycoprotein with adhesion functions (Hughes et al., 1994), and insulin-like growth factor 1 (Lean et al., 1995). Fifty percent of osteocytes in situ express estrogen receptor (Braidman et al., 1995), and avian osteocytes appear to express specific antigens detected by a monoclonal antibody not expressed on avian osteoblasts (Nijweide and Mulder, 1986). It is generally accepted that osteocytes are low expressors of alkaline phosphatase and recently it has been shown that osteocytes produce greater amounts of casein kinase activity compared with osteoblasts (Mikuni-Takagaki et al., 1995). It is very likely that mammalian osteocytes produce markers distinctly different from those of osteoblasts.
Arden and co-workers (1994) have stated that for the osteocyte to survive, the cell must maintain an unmineralized area around the body of the cell and around the cell processes. This is necessary in order to allow the diffusion of nutrients and waste products to and from the cell. Mikuni-Takagaki and co-workers (1995) described the extracellular accumulation of a large amount of osteocalcin around isolated osteocytes. Osteocalcin has been described in the endoplasmic reticulum and Golgi cisternae in osteocytes (Ohta et al., 1989; Boivin et al., 1990). Recently, Ducy and co-workers (1996) have demonstrated that mice which lack the functional gene for osteocalcin have increased cortical and trabecular bone which lead them to postulate that osteocalcin is an inhibitor or negative regulator of mineralization. The osteocyte may produce large amounts of osteocalcin to prevent the mineral from closing off the cell body and processes.
It has been hypothesized that osteocytes respond to loading pressures on bone by signaling osteoblasts to produce new bone (for review, see Burger et al., 1993). Recently it has been shown that loaded bone contains fewer apoptotic osteocytes (Noble et al., 1997) and that osteocyte cell death is increased during estrogen withdrawal (Tomkinson et al., 1996) and during treatment with excess glucocorticoid (Weinstein et al., 1997) suggesting that bone loss or bone necrosis is due to osteocyte death which prevents normal bone remodeling or normal bone repair. If osteocytes are the cell responsible for sensing mechanical stress and for signaling osteoblasts to produce new bone, then understanding their functions could lead to new therapies to prevent or restore bone loss due to immobilization or other processes.
The study of osteocytes has utilized immunohistochemistry techniques and the isolation of primary cells. However, primary cells can only be obtained in relatively low numbers and in heterogeneous populations. An osteocyte cell line would prove useful to study the properties of osteocytes through the use of molecular and functional techniques which require relatively large numbers of homogeneous cells.
We postulated that since osteocytes are large producers of osteocalcin, that bone cells derived from transgenic mice overexpressing the T-antigen driven by the osteocalcin promoter which would serve to target large T-antigen to osteoblasts and osteocytes, and thereby be a source of immortalized cells of these types. We chose to use cellular morphology as the initial criteria for cloning cell lines with osteocytes characteristics from isolates from these mice (Chen et al., 1995). Once clonal cell lines were established, they were characterized as far as the osteocyte/osteoblast phenotypes were concerned.
With regard to the generation of monoclonal antibodies specific for osteocytes, Nijweide and co-workers reported a monoclonal antibody which recognizes avian osteocytes but not mammalian osteocytes (Nijweide and Mulder, 1986). This monoclonal antibody was generated by injecting mice with osteoblast-like cells derived from digestions of chick embryol calvaria which had been cultured 6 days before injection. This monoclonal antibody specifically reacts with the cell surface of osteocytes and not with any specific band by western blotting of chick osteocyte lysate. The specificity of this monoclonal antibody has been confirmed by Bruder and Caplan (1989) and this antibody has been used as a tool to purify osteocytes (Vanderplas et al., 1994) and investigate osteocyte function (Tanaka et al., 1995).
Another monoclonal antibody which recognizes the osteoblast to osteocyte transition has been generated. This antibody recognizes a cell surface antigen called E11 in rats which is homologous to the OTS-8/ap38 molecule in mice (Wetterwald et al., 1996). This monoclonal antibody was generated by injecting mice with the rat osteoblastic cell line IRC 10/30-myc3. The E11 transcript was detected in bone, lung, brain, and skin. This antigen appears to be expressed during the transition stage from the osteoblast to the osteocyte phenotype. Over-expression of E11 in ROS 17/2.8 cells caused these cells to form long cytoplasmic extensions (Sprague et al., 1996). Therefore this antigen appears to be an osteocyte differentiation agent.
We were successful in generating monoclonal antibodies which are specific for mammalian osteocytes, by immunizing a rat with the cultured osteocytes originated from the osteocyte cell lines of the current invention.
These cell lines and monoclonal antibodies should prove to be useful tools to examine the functions of osteocytes as a whole as well as to characterize osteocyte specific antigens and their roles in osteocyte function.
References related to the invention as cited above and also references hereinafter are listed herein below:
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Establishment of an osteocyte cell line would make possible studies using molecular and functional techniques which require large numbers of cells in a homogeneous population. First, monoclonal antibodies could be developed which specifically recognize osteocytes. These antibodies could be used for immunocytochemistry and affinity purification of primary osteocytes as well as characterization of osteocyte-specific antigens and their role in osteocyte function. Secondly, these cells could be used to examine the effects of mechanical stress as potential signaling factors may be released by these cells. Molecular techniques could be used to examine regulation of messenger RNA and subtraction techniques could be utilized to determine which factors are induced by mechanical stress. Thirdly, these cells could be used to determine cell-cell communication between osteocyte-osteocyte, osteocyte-osteoblast, and even osteocyte-osteoclast. Expression of connexins and gap junction proteins could be examined. Fourthly, these cells could be used to examine the effects of unknown compounds on the functional characteristics of osteocytes to develop potential new therapies to induce new bone formation. Fifthly, these cells could be used to screen for modification factor of the osteocyte cell line by monitoring a material such as NO, NOS activity, prostaglandins, COX activity, osteocalcin, IGFs, TGF-beta, connexins, kinase activity, Ca2+ uptake, ion channel activity and 3[H]-thymidine uptake. Lastly, these cells could be used to screen for factors that bind to the osteocyte.
We describe herein the establishment of several cell lines with the characteristics of osteocytes derived from transgenic mice which overexpress the T-antigen driven by the osteocalcin promoter. These cell lines were characterized and their properties compared with the known properties of primary osteocytes, osteoblasts, and other cells. They display various degrees of dendritic processes, are high producers of osteocalcin and osteopontin. In certain mature osteocyte cell lines, connexin 43 is expressed in high levels. Both control osteoblast cell line and the primary osteocytes cell line express CD44, therefore CD44 is not a specific marker for osteocytes. Furthermore, several osteocyte cell lines formed mineral on their cell surface. Most interestingly, one of the cell lines established, MLO-Y4 for xe2x80x98murine long bone osteocytexe2x80x99 has properties that are very similar to primary osteocytes. Like primary osteocytes and unlike primary osteoblasts, the cell line produces large amounts of osteocalcin but low amounts of alkaline phosphatase. The cells produce extensive, complex dendritic processes, are positive for T-antigen, for osteopontin, for the neural antigen CD44, and for connexin 43, a protein found in gap junctions. This cell line also produces very small amounts of type I collagen mRNA compared with primary osteoblasts. MLO-Y4 cells lack detectable mRNA for osteoblast-specific factor 2 (OSF-2) which appears to be a positive marker for osteoblasts but may be a negative marker for osteocytes. OSF-2 is highly expressed in primary osteoblasts and MC3T3-E1 cells, but not apparently in osteocytes. The cloned dendritic cell lines may represent various stages of differentiation of the osteocyte.
We also describe herein the generation of monoclonal antibodies which specifically recognize osteocyte-specific antigens expressed at different sites and/or different stages of osteocytes.
Accordingly, it is an object of present invention to provide a method of producing osteocyte cell line in various stages of differentiation. Such osteocyte cell line remains stable after more than 20 passages. The osteocyte has a stellate shape with dendritic processes and expresses high levels of osteocalcin.
Another object is to provide a method of production for cultured osteocyte which remains stable after more than 20 passages. The osteocyte has a stellate shape with dendritic processes and expresses high levels of osteocalcin.
Another object is to provide osteocyte cell line of a differentiation stage which remains stable after more than 20 passages. The osteocyte has a stellate shape with dendritic processes and expresses high levels of osteocalcin.
Yet another object is to provide a cultured osteocyte which remains stable after more than 20 passages. The osteocyte has a stellate shape with dendritic processes and expresses high levels of osteocalcin.
Yet another object is to provide a method of producing monoclonal antibodies which specifically recognize osteocyte-specific antigens.
Yet another object is to provide monoclonal antibodies which specifically recognize osteocyte-specific antigens.
Yet another object is to provide a method of screening for modification factors of the osteocyte cell line.
Yet another object is to provide a method of screening for binding factors with the osteocyte.
These and other objects of the invention as well as a fuller understanding of the advantages thereof, can be had by reference to the following description and claims.
The invention provides a method of producing osteocyte cell line in various stages of differentiation. Such cell line remains stable after more than 20 passages. The osteocyte has a stellate shape with dendritic processes and expresses high levels of osteocalcin. The method comprises the steps of preparing a transgenic animal carrying an osteocalcin promoter driven T-antigen transgene. Bones are then isolated from the transgenic animal and digested with collagenase solution. The cells are then harvested into fetal and adult calf serum supplemented medium. The harvested cells are then plated and a cell line is isolated by selecting single colony.
The invention also provides a method of producing osteocyte cell line in various stages of differentiation. Such cell line remains stable after more than 20 passages. The osteocyte has a stellate shape with dendritic processes and expresses high levels of osteocalcin. The method comprises the steps of preparing a transgenic animal carrying an osteocalcin promoter driven T-antigen transgene. Bones are then isolated from the transgenic animal and digested with collagenase solution. The remaining bone pieces are then harvested and alternately treated with EDTA and collagenase. The remaining bone pieces are then minced into smaller chips. The bone chips are then cultured for a period sufficient to allow migration of cells from the bone chips. The migrated cells are then harvested and cultured with fetal and adult calf serum supplemented medium. A cell line is then isolated by selecting single colony.
The invention further provides a method of producing cultured osteocyte. The cultured osteocyte remains stable after more than 20 passages. It has a stellate shape with dendritic processes and expresses high levels of osteocalcin. The method comprises the steps of preparing a transgenic animal carrying an osteocalcin promoter driven T-antigen transgene. The bones are then isolated from the transgenic animal and digested with collagenase solution. The cells are then harvested and cultured with fetal and adult calf serum supplemented medium.
The invention yet further provides a method of producing cultured osteocytes. The cultured osteocyte remains stable after more than 20 passages. The osteocyte has a stellate shape with dendritic processes and expresses high levels of osteocalcin. The method comprises the steps of preparing a transgenic animal carrying an osteocalcin promoter driven T-antigen transgene. The bones are then isolated from the transgenic animal and digested with collagenase solution. The remaining bone pieces are then harvested and alternately treated with EDTA and collagenase. The remaining bone pieces are then minced into smaller chips. The bone chips are then cultured for a period sufficient to allow migration of cells from bone chips. The migrated cells are then harvested and cultured with fetal and adult calf serum supplemented medium.
Yet the invention further relates to a osteocyte cell line in various stages of differentiation. The osteocyte cell line remains stable after more than 20 passages. The osteocyte has a stellate shape with dendritic processes and expresses high levels of osteocalcin.
Yet the invention further relates to a cultured osteocyte. The cultured osteocyte remains stable after more than 20 passages. The osteocyte has a stellate shape with dendritic processes and expresses high levels of osteocalcin.
Yet the invention further provides a method of producing monoclonal antibodies which specifically recognize osteocyte-specific antigens. The method comprises the steps of immunizing an animal with cultured osteocytes prepared by the current invention as described above, obtaining antibody-producing cells from the immunized animal, forming hybridomas by fusing antibody-producing cells with immortalizing cells and harvesting the monoclonal antibodies produced by the hybridomas.
Yet the invention further provides monoclonal antibodies which specifically recognize osteocyte-specific antigens.
Yet the invention further provides a method of screening for modification factors of the osteocyte cell line by monitoring a material such as NO, NOS activity, prostaglandins, COX activity, osteocalcin, IGFs, TGF-beta, connexins, kinase activity, Ca2+ uptake ion channel activity and 3[H]-thymidine uptake.
Yet the invention further provides a method of screening for factors that binds to osteocytes.